WO2022126685A1

WO2022126685A1 - System and method for in vivo observation of intraosseous capillary in animal body

Info

Publication number: WO2022126685A1
Application number: PCT/CN2020/138080
Authority: WO
Inventors: 赵莹子; 李泊泰; 巨富荣; 高大双; 杨帆
Original assignee: 中国科学院深圳先进技术研究院
Priority date: 2020-12-18
Filing date: 2020-12-21
Publication date: 2022-06-23
Also published as: CN114642401A

Abstract

A system and method for in vivo observation of an intraosseous capillary in an animal body. The system comprises a fixing device (2), a polishing device, and a two-photon microscope (1). The fixing device (2) is connected to an objective table (11) of the two-photon microscope (1) and configured to fix an animal body to be observed; the polishing device is configured to polish an exposed bone of said animal body to thin bone cortex; and the two-photon microscope (1) is configured to image the polished bone part to observe a capillary therein, wherein the fixing device (2) comprises a bearing substrate (21), two or more support arms (22) connected on the bearing substrate (21), and clamping arms (23) detachably assembled on the support arms (22); and a locking member (24) is provided on the support arm (22) and configured to lock the clamping arms (23) assembled on the support arms (22). According to the system and the method, stable imaging of intraosseous capillaries in living animals can be realized, and abundant intraosseous capillary morphology is observed in real time in vivo.

Description

System and method for in vivo observation of capillaries in animal bone

technical field

The invention belongs to the technical field of medical imaging, and in particular relates to a system for in vivo observation of capillaries in animal body bones and a corresponding method.

Background technique

Vascular endothelial cells are monolayer cells distributed in the inner wall of blood vessels, with rich heterogeneity and organ specificity. Through dense capillary branches, the vascular endothelium establishes connections with nearly all cells in various organs. Vascular endothelial cells not only serve as a pipeline system to transport nutrients and take away metabolic wastes, but also play an important role in the growth, development and homeostasis of various organs by secreting vascular secretion factors. Capillaries in different organs have different shapes, and capillaries in different locations and functions in the same organ have different shapes, which reflect the characteristics of different subtypes of endothelial cells. With the occurrence of age or disease, its morphology will also change, and its homeostasis maintenance effect on organs will also change accordingly. Therefore, observing the morphology of capillaries can provide information on the state of the corresponding organ.

There are two ways to observe the morphology of capillaries in vitro and in vivo. Due to the deep position of capillaries in the bone, the current technology is mainly based on in vitro observation. For example, after decalcification of the isolated bone, the bone is cut into slices ranging from 30 microns to 300 microns, and the morphology of the capillaries is observed by light microscope or confocal microscope after specific fluorescent staining of capillaries. For another example, the isolated bone is made transparent after chemical treatment, and then the blood vessels are specifically fluorescently stained, and then photographed by a surface-selective illumination fluorescence microscope, and the image of capillaries is obtained by digital processing in the later stage. Existing in vitro observation of capillary morphology in bone limits its application range, and it is necessary to explore in vivo observation methods in order to obtain more abundant real-time observation results.

SUMMARY OF THE INVENTION

In view of the deficiencies in the prior art, the present invention provides a system and method for in vivo observation of capillaries in bones of animals, so as to achieve stable imaging of capillaries in bones in vivo.

In order to achieve the above object of the invention, one aspect of the present invention is to provide a system for in vivo observation of capillaries in animal bones, the system includes a fixation device, a polishing device and a two-photon microscope; the fixation device is connected to the On the stage of the two-photon microscope, it is used to fix the animal body to be observed; the grinding device is used to grind the exposed bones of the animal body to be observed to thin the cortical bone; the two-photon microscope is used for grinding Image the polished bone to see the capillaries in it;

Wherein, the fixing device includes a carrier substrate, two or more supporting arms connected to the carrier substrate, and a clamping arm detachably assembled on the supporting arms, and a locking member is provided on the supporting arms , used to lock the clamping arm assembled on the support arm.

Preferably, a groove is formed on the top of the support arm, and the clamping arm is fitted into the groove.

Preferably, the number of the support arms is two or three, and each of the support arms is respectively provided with one of the clamping arms.

Preferably, the carrier substrate is provided with elongated through holes.

Preferably, one end of the elongated through hole communicates with the edge of the carrier substrate.

Preferably, the support arm is configured as a height-adjustable support arm.

Preferably, the support arm is configured as a support arm movable on the plane of the carrier substrate.

Another aspect of the present invention is to provide a method for in vivo observation of capillaries in animal bones, using the system for in vivo observation of capillaries in animal bones to observe capillaries, the method comprising:

Fixing the fixing device on the stage of the two-photon microscope;

Place the animal object to be observed between two or more support arms of the fixing device, control the clamping arm to clamp the animal object to be observed on the carrier substrate and lock the clamp arm;

After anesthetizing the body of the animal to be observed, the target observation area is depilated, and then the skin and muscles are cut to expose the bones of the target observation area;

grinding the exposed bone with the grinding device to thin the cortical bone;

An vascular imaging agent is injected into the animal to be observed, and then the two-photon microscope is activated to image the polished bone to observe the capillaries therein.

The system and method for in vivo observation of capillaries in animal bones provided by the embodiments of the present invention utilize the characteristics of deep imaging with two-photon microscopy, less damage to living cells, and longer imaging time to observe intrabony capillaries in vivo, The imaging position is deep and the imaging is stable; the animal body to be observed is fixed by designing a fixing device with a specific structure, which overcomes the problem of image displacement caused by the breathing and heartbeat of the living body, and makes the imaging more stable; the exposed bone to be observed is polished and thinned by using a polishing device The cortical bone increases the depth of the imaging position, and can image and observe the capillaries in the deeper position.

Description of drawings

1 is a schematic structural diagram of a system for in vivo observation of capillaries in animal body bones according to an embodiment of the present invention;

Figures 2 and 3 are illustrations of intraosseous capillary imaging of experimental mice in an embodiment of the present invention.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Examples of these preferred embodiments are illustrated in the accompanying drawings. The embodiments of the invention shown in the drawings and described with reference to the drawings are merely exemplary and the invention is not limited to these embodiments.

Here, it should also be noted that, in order to avoid obscuring the present invention due to unnecessary details, only the structures and/or processing steps closely related to the solution according to the present invention are shown in the drawings, and the related structures and/or processing steps are omitted. Other details not relevant to the invention.

The embodiment of the present invention first provides a system for observing capillaries in animal body bone in vivo, referring to FIG. The two-photon microscope 1 includes a stage 11 and an imaging lens 12. The fixing device 1 is connected to the stage 11 of the two-photon microscope 1, and is used to fix the object to be observed. The imaging lens 12 of the microscope 1 is used to image the polished bone to observe the capillaries therein. The grinding device is used for grinding the exposed bones of the animal body to be observed, so as to thin the cortex of the bone, so as to increase the depth of the imaging position, and the intraosseous capillaries in the deeper position can be imaged and observed.

Wherein, as shown in FIG. 1 , the fixing device 2 includes a carrier substrate 21 , two or more support arms 22 connected to the carrier substrate 21 , and a clamping arm detachably assembled on the support arms 22 . 23 , the support arm 22 is provided with a locking member 24 for locking the clamping arm 23 assembled on the support arm 22 .

In a preferred solution, the number of the support arms 22 is two or three, and each of the support arms 22 is configured with one of the clamping arms 23 respectively. The fixation device 2 shown in FIG. 1 comprises three support arms 22, and the area between the three support arms 22 is formed as a space for placing the animal body to be observed.

In a preferred solution, the support arm 22 is configured as a height-adjustable support arm, so that the height of the space for placing the animal object to be observed is adjustable, so as to be suitable for animal objects of different sizes.

In a further preferred solution, the support arm 22 is configured as a movable support arm on the plane of the carrier substrate 21, so that the lateral width for placing the animal to be observed is adjustable to suit different body sizes. animal body.

In a preferred solution, as shown in FIG. 1 , a groove 25 is formed on the top of the support arm 22 , the clamping arm 23 is assembled in the groove 25 , and the locking member 24 is set as a thread locking piece.

In a preferred solution, as shown in FIG. 1 , an elongated through hole 26 is formed on the carrier substrate 21 , and the fixing device 2 can be fixedly connected through the elongated through hole 26 and a threaded fastener. On the stage 11 of the two-photon microscope 1, it is arranged as a long strip through hole so that the connection position can be easily adjusted. In a further preferred solution, as shown in FIG. 1 , one end of the elongated through hole 26 communicates with the edge of the carrier substrate 21 .

Another aspect of the present invention is to provide a method for in vivo observation of capillaries in animal bones, using the system for in vivo observation of capillaries in animal bones to observe capillaries. In the embodiment of the present invention, the animal body to be observed is an experimental mouse as an example for description. In conjunction with the schematic structural diagram of Fig. 1, the method includes the following steps:

(1), the fixing device 2 is fixedly connected to the stage 11 of the two-photon microscope 1 .

(2) Place the experimental mouse (not shown in FIG. 1 ) between the support arms 22 of the fixing device 2 , and control the clamping arm 23 to clamp the animal to be observed on the The clamping arm 23 is mounted on the carrier substrate 21 and locked.

(3) After the experimental mice were anesthetized, depilatory treatment was performed on the thigh bones in the target observation area, and then the skin and muscles were cut to expose the bones in the target observation area.

(4), using the grinding device to grind the exposed bone to thin the cortical bone. For example, a micro-drill is used to gently remove part of the cortical bone, with the limit of breaking the vascular structure inside the bone without grinding bleeding.

(5), inject an angiographic imaging agent into the experimental mice. For example, TRITC-labeled dextran (TRITC-Dextran, average molecular weight 65000-85000, final injection concentration 0.1 mg/kg) was injected from the orbit of the mouse.

(6), start the two-photon microscope to image the polished bone, and observe the capillaries therein.

Further, after the imaging, the muscle and skin tissues of the experimental mice were sutured, so that the mice could survive after recovery from anesthesia, and the same mice could be used to repeat the related experiments afterwards.

FIG. 2 and FIG. 3 are illustrations of intraosseous capillary imaging of the thigh region of experimental mice in an embodiment of the present invention. Among them, Figure 2 shows the capillaries of the arched column structure near the growth plate (gp) of the upper epiphysis (mp), and Figure 3 shows the capillaries of the diaphyseal end (dp) network structure. It can be known from FIG. 2 and FIG. 3 that the system and method provided by the embodiments of the present invention can realize in vivo observation of capillaries of different shapes in animal body bones. Two different forms of capillary endothelial cells have different physiological effects on bone tissue: vascular endothelial cells in the arched columnar structure near the epiphyseal (mp) growth plate (gp) mainly promote bone formation, while the diaphyseal (dp) reticular Structural blood vessels have a regulatory effect on hematopoiesis of bone marrow cells. Therefore, by distinguishing different shapes of intraosseous capillaries, the heterogeneity of capillary endothelial cells can be judged and their physiological state can be judged.

To sum up, the system and method for in vivo observation of capillaries in animal bones provided by the embodiments of the present invention utilize the characteristics of deep imaging with two-photon microscopy, less damage to living cells, and longer imaging capability to observe living cells. Intraosseous capillaries, the imaging position is deeply incised, and the imaging is stable; by designing a fixed device with a specific structure to fix the animal body to be observed, the problem of image displacement caused by living breathing and heartbeat is overcome, and the imaging is more stable; the exposure to be observed is exposed by using a polishing device The bone is polished to thin the cortical bone, which increases the depth of the imaging position and enables imaging and observation of the capillaries in the bone at a deeper position.

The above are only specific embodiments of the present application. It should be pointed out that for those skilled in the art, without departing from the principles of the present application, several improvements and modifications can also be made. It should be regarded as the protection scope of this application.

Claims

A system for in vivo observation of capillaries in animal bones, wherein the system comprises a fixing device, a polishing device and a two-photon microscope; the fixing device is connected to the stage of the two-photon microscope, and is used for treating The observed animal body is fixed; the grinding device is used for grinding the exposed bones of the animal body to be observed, so as to thin the cortical bone; the two-photon microscope is used for imaging the polished bone part to observe the the capillaries;

Wherein, the fixing device includes a carrier substrate, two or more supporting arms connected to the carrier substrate, and a clamping arm detachably assembled on the supporting arms, and a locking member is provided on the supporting arms , used to lock the clamping arm assembled on the support arm.
The system for in vivo observation of capillaries in animal body bones according to claim 1, wherein a groove is formed on the top of the support arm, and the clamping arm is fitted in the groove.
The system for in vivo observation of capillaries in animal body bones according to claim 1, wherein the number of the support arms is two or three, and each of the support arms is respectively provided with one of the clamping arms.
The system for in vivo observation of capillaries in animal body bones according to claim 2, wherein the number of the support arms is two or three, and each of the support arms is respectively provided with one of the clamping arms.
The system for in vivo observation of capillaries in animal body bones according to claim 1, wherein elongated through holes are formed on the carrier substrate.
The system for in vivo observation of capillaries in animal bones according to claim 5, wherein one end of the elongated through hole communicates with the edge of the carrier substrate.
The system for in vivo observation of capillaries in animal body bones according to claim 1, wherein the support arm is configured as a height-adjustable support arm.
The system for in vivo observation of capillaries in animal body bone according to claim 7, wherein the support arm is configured as a support arm movable on the plane of the carrier substrate.
The system for in vivo observation of capillaries in animal bones according to claim 2, wherein the support arm is configured as a height-adjustable support arm, and the support arm is further configured to be adjustable on the plane of the carrier substrate Mobile support arm.
A method for in vivo observation of capillaries in animal bones, wherein the capillaries are observed by using the system for in vivo observation of capillaries in animal bones according to any one of claims 1-9, the method comprising:

Fixing the fixing device on the stage of the two-photon microscope;

Place the animal object to be observed between two or more support arms of the fixing device, control the clamping arm to clamp the animal object to be observed on the carrier substrate and lock the clamp arm;

After the body of the animal to be observed is anesthetized, the target observation area is depilated, and then the skin and muscles are cut to expose the bones of the target observation area;

grinding the exposed bone with the grinding device to thin the cortical bone;

An vascular imaging agent is injected into the animal to be observed, and then the two-photon microscope is activated to image the polished bone to observe the capillaries therein.